PROJECT SUMMARY The spatial organization of DNA within the nucleus is critical for proper gene expression and cellular function. Patterns of genome folding can vary by cell type and are perturbed in human diseases such as developmental syndromes and cancers. The molecular mechanisms that govern genome organization are poorly understood and yet are critically important for human health. A hierarchy of structures link genome topology and activity. Critical structures for gene control are the chromatin loops that bring genes and their regulatory elements together in close physical proximity. Genome- wide profiling indicates that there are more than 10 regulatory elements for every gene, yet little is known about how regulatory elements find their target genes or how they function at the molecular level. A major goal in the field is to identify all of the DNA loops in the genome and determine how they act individually and in combination to regulate gene expression. The Structural Maintenance of Chromosome (SMC) complexes are a family of proteins that play key roles in shaping the three-dimensional architecture of the genome. The two major SMC complexes, Cohesin and Condensin, were first identified for their roles in chromosome re-organization during the cell cycle. Recent work has implicated these factors in gene regulation during interphase, and the assumption is that these ring- shaped protein complexes act by facilitating loops in DNA. It is important to uncover the mechanisms that determine where and how Cohesin and Condensin interact with the genome and the functional consequences of these loop structures to development and disease processes. The long-term scope of this research program is to move from a linear view of the genome to a panoramic view where the physical orientation of the genome in three-dimensional space directs gene expression. This project will focus on three major questions. First, what does each architectural protein contribute to the overall topology of the genome? Second, how are architectural proteins regulated during DNA looping and gene regulation? Third, how do DNA loops impact gene activity? These studies will assess the biochemical and molecular processes that control gene expression and DNA looping and determine the consequences of specific mutant architectural proteins. This research will shed light on how regulatory elements control genome organization, direct gene expression, and define cell types during development.